Antarctic fungi with antibiotic potential isolated from Fort William Point, Antarctica

The Antarctic continent is one of the most inhospitable places on earth, where living creatures, mostly represented by microorganisms, have specific physiological characteristics that allow them to adapt to the extreme environmental conditions. These physiological adaptations can result in the production of unique secondary metabolites with potential biotechnological applications. The current study presents a genetic and antibacterial characterization of four Antarctic fungi isolated from soil samples collected in Pedro Vicente Maldonado Scientific Station, at Fort William Point, Greenwich Island, Antarctica. Based on the sequences of the internal transcribed spacer (ITS) region, the fungi were identified as Antarctomyces sp., Thelebolus sp., Penicillium sp., and Cryptococcus gilvescens. The antibacterial activity was assessed against four clinical bacterial strains: Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, and Staphylococcus aureus, by a modified bacterial growth inhibition assay on agar plates. Results showed that C. gilvescens and Penicillium sp. have potential antibiotic activity against all bacterial strains. Interestingly, Thelebolus sp. showed potential antibiotic activity only against E. coli. In contrast, Antarctomyces sp. did not show antibiotic activity against any of the bacteria tested under our experimental conditions. This study highlights the importance of conservation of Antarctica as a source of metabolites with important biomedical applications.


Results
Fungi phylogeny. The phylogenetic analysis of the ITS sequences generated in this study (MZ958929, MZ958928, MZ958926, MZ958927) revealed that the T4-400-5E, T4-1K-1A and T4-1K-1G isolates clustered with the phylum Ascomycota, identified as Penicillium sp., Antarctomyces sp. and Thelebolus sp., respectively. The isolate T4-200-3B, clustered with the phylum Basidiomycete, was identified as Cryptococcus gilvescens (Fig. 1). All clades were strongly supported by bootstrap values higher than 70%. The tree grouped the family Thelebolaceae (Thelebolus and Antarctomyces genera) in a monophyletic group with a bootstrap value of 99% and segregated the family Trichocomaceae (Penicillium genus) in a separate group with a high 100% bootstrap value. Cryptococcus gilvescens was described as a more genetically distant species and the sequences contemplated in the tree were further divided into two additional clades with bootstraps well supported and within the family Tremellaceae.
Morphological observations. On PDA media the macro and microscopic morphological identification of the fungi isolates corroborated the topology of the analyzed genetic sequences.
Penicillium sp. (T4-400-5E) showed a yellow coloration on the agar. The colony had a furrowed appearance surrounded by a white margin. The center was umbonated with blue-green coloration. On the reverse, the colony had a yellow coloration. After 20 and 30 days of growth, the colony had a diameter of 2 cm and 2.5 cm, respectively (Fig. 2e,f). Microscopically, the anamorphic structure was represented with monoverticillate penicillin, the stipes were septated and the phialides measured 9 × 3 µm. The conidia were ovoid, with a dimension of 3.5 × 3 µm (Fig. 3c).
Cryptococcus gilvescens (T4-200-3B) presented a cream yeast-like colony with mucoid texture. The colony had a diameter between 1 and 1.7 cm at 15 and 30 days of growth, respectively (Fig. 2g,h). Microscopically, the cells were round to oval, with a diameter of 2.5-3 µm. Asexual reproduction by budding was observed (Fig. 3d).

Antibiotic susceptibility tests. Standard antibiotic resistant tests with the four bacteria used in this study
showed that S. aureus was susceptibility to all the antibiotics tested, whereas K. pneumoniae, E. coli, and E. faecalis showed resistant to three or more antibiotics (Supplementary file 1).
The modified antibacterial susceptibility test performed on K. pneumoniae with the PDA and temperature treatment showed that the bacteria was still resistant to imipenem (imp) and meropenem (mem) regardless of the growth conditions (Supplementary file 2). Although a slight ring of growth inhibition was noticed on PDA as compared to LB agar, it was considered still a resistant phenotype.
Antibacterial potential. The antibacterial potential of the fungi was determined by the observation of bacterial growth inhibition zone around the mycelia plug (Fig. 4). Inhibition by Thelebolus sp. was only observed in the bioassay with E. coli at 15, 30 and 60 days of growth (Fig. 4a). At three different times of growth, C. gilvescens showed inhibitory effects to all Gram-positive and Gram-negative bacteria tested (Fig. 4b). Antarctomyces sp. did not show antibacterial activity against all the bacteria tested (Fig. 4c). Similar to C. gilvescens, Penicillium sp. showed inhibitory effects to all Gram-positive and Gram-negative bacteria tested at 15 and 30 days of growth (Fig. 4d).
In order to determine if growth time of the fungi (i.e. 15, 30 and 60 days) had a significant effect on the observed inhibition halo size, we conducted a Kruskal-Wallis and a Mann-Whitney statistical test to determine the difference between three and two different growth times, respectively. www.nature.com/scientificreports/ Statistical analysis on the size of bacterial growth inhibition ring around mycelia plugs, previously incubated at 15, 30, and 60 days, before being exposed to the bacterial lawns, showed significant differences (p < 0.05) with E. coli and Thelebolus sp. plugs. On the other hand, C. gilvescens showed similar growth inhibition rings at all three growing periods for the four bacteria tested in the assay (p > 0.05). Similar results were obtained with bacteria exposed to 15 and 30 days grown of Penicillium sp. plugs (p > 0.05) (Supplementary file 3). Antarctomyces sp. was not included because it did not show antibacterial properties in our assays.

Discussion
The kingdom Fungi is considered a key contributor to the biotechnology industry 42 , with several applications in textile, food, and pharmaceutics processes 43,44 . Valuable compounds with antitumor, antiparasitic and antibacterial activity have been identified in fungi from Antarctica [45][46][47][48][49][50] . Although they have great potential as novel source of compounds, the genetic diversity of microbes from this pristine and unique polar environment is largely unexplored 51 . In this study, we describe the genetic and morphological characterization of four soil fungi isolated from Fort William Point, Greenwich Island, near Pedro Vicente Maldonado Ecuadorian Antarctic Research Station, some of which showed bioactivity against relevant clinical bacterial isolates.
For the genetic characterization of our selected isolates, we used a phylogenetic tree based on the sequence of the ITS region, which is considered as the barcode for fungal taxonomy [52][53][54] . There is some disagreement whether this region alone has enough variability as a reliable species-specific identification marker [55][56][57][58][59] . This has been shown to be the case with some genera in the Ascomycota 60 . Some authors suggest that using the ITS region along with other protein-coding genes such as RPB1 (RNA polymerase II largest subunit, regions E and F), RPB2 (RNA polymerase II second largest subunit, regions 5-7), Tsr1 (20S pre-rRNA processing protein), Cct8 (subunit of the cytosolic chaperonin Cct ring complex) and MCM7 (Minichromosome Maintenance Complex Component 7) to identify fungal species of the same genera with low intraspecific variation 55 . ITS region together with other genes such as calmodulin and β-tubulin have been useful in deeper taxonomical studies to discriminate between the genera Penicillium 58 , which has proven difficult to classify among the fungi taxa 55 . Recently, the ITS combined with fragments of β-tubulin and RPB2 were successfully used to identify a new species of Antarctomyces 61 , and to differentiate closely related fungi with low genetic variation 62 . However, other studies described β-tubulins as phylogenetically misleading, because they are present in the genome in multiple copies 63,64 . Species delimitation remains a challenging issue for closely related and cryptic fungal species 65,66 , and additional barcode markers, other than ITS, are being developed 59 .
In our study, we successfully confirmed the genus of our selected fungi with the use of a phylogenetic tree based on ITS sequencing. Isolates related to the Ascomycota group were confirmed as Penicillium sp., Thelebolus sp., and Antarctomyces sp. Identification to the species level for this group can be achieved with the implementation of additional gene sequence in upcoming studies. For a single isolate, the ITS sequencing allowed for species identification of the isolate T4-200-3B as C. gilvescens. Fungal morphological structures observed in this study were similar to previously descriptions for the same genera 67 . The integration of molecular data with other classification techniques such as morphology, ecology, new generation sequencing, and chemical profiling is nowadays our best set of tools to achieve a successful characterization of the fungi 61,68-71 .
Additionally, our phylogenetic analysis clearly separated the species of the Basidiomycota and Ascomycota phyla. Antarctomyces sp. and Thelebolus sp. segregated into sister clades that share an immediate common ancestor. These cryophilic genera have a slow generation time and thus accumulate only minor mutations, evolving slower than other species 72 . According to their geographic distribution, Thelebolus genus is known for its cryophilic nature and for its association with dung and guano 72 . Some species such as T. globosus and T. ellipsoideus are endemic to Antarctica, while others such as T. microsporus have a wider habitat, including Antarctica 16,72-74 . The genus Antarctomyces includes only two species, both native to the Antarctic continent 16,61,75 . Sharing the same phylum, Penicillium sp. clustered within the P. lividum and P. odoratum clade, and showed a strong bootstrap value with other species of the genus; all belonging to the section Aspergilloides 60,76 . The Basidiomycota phylum is represented by Cryptococcus gilvescens. This species distribution is restricted to cold environments, including the Antarctica 77,78 , where it is considered the most abundant genus of yeast 79 . C. gilvescens also showed a close relationship with C. gastricus, as previously reported 78 .
Bioactivity potential against pathogens is a promising application of the genetically diverse fungi of Antarctica. For instance, C. gilvescens and Penicillium sp. have shown antibiotic potential against Gram-negative bacteria, such as E. coli and K. pneumoniae, and Gram-positive bacteria, such as E. faecalis and S. aureus. This agrees in part with previous reports on the antibacterial activity of Cryptococcus species against Gram-positive bacteria 22,80 . In our study, C. gilvescens also showed antibacterial potential for Gram-negative bacteria. Additionally, C. gilvescens was reported to express extracellular lipolytic/esterasic activity, starch-degrading activity 81 , extracellular amylase, lipase, and protease activities 78 , anti-yeast activity 82 , and laccasse activity 83 . Various Cryptococcus isolated from Antarctic marine sediments had also exhibited lipase, esterase, and pectinase activity 84 .
In relation to species of Penicillium isolated from diverse polar ecosystems, such as marine sediments, deepsea sediments, and sea-bed sediments, it is known that this genus has cytotoxic effects against cancer cell lines, anti-inflammatory effect, anti-allergic effect, antifungal and antibacterial activities 84 . A novel strain of Penicillium found in Antarctic soil showed production of three new indolyl diketopiperazine derivatives and seven known alkaloid compounds 85 . Some of these compounds had significant in vitro cytotoxic activity against cancer cell lines and one of them had antituberculosis activity 85 . An early study described nephrotoxicity in humans and strong antibiotic activity with P. odoratum 86 . This fungus produces the hazardous citrinin toxin, a mycotoxin that causes nephrotoxicity in humans [87][88][89] . Because the citrinin gene appears to be highly conserved within the genus Penicillium 90 , it is likely that citrinin is present in our Penicillium sp. isolate. P. lividum presented cytotoxic www.nature.com/scientificreports/ activity associated with the production of meroterpenoid compounds 91 . Furthermore, we report a Penicillium strain (Penicillium sp.) that produced antibacterial activity against Gram-negative and Gram-positive bacteria.
Previous studies have documented antitumoral 20 and antibiotic potential in the Thelebolus genus, although the latter was less potent than Penicillium 35 . Thelebolus sp. from the Himalayas showed no antimicrobial activity against Gram-negative bacteria, but did exhibit antimicrobial activity against Gram-positive bacteria 92 . In contrast, Thelebolus sp. isolated in this study showed antibacterial activity against the Gram-negative bacteria E. coli. Several biotechnological applications have been attributed to T. microsporus due to the synthesis of linolenic acid, carotenoid pigments and extracellular α-amylase activity 93 . Lastly, our Antarctomyces isolate did not show any antibacterial activity against the tested bacteria in our in vitro assay conditions. Members of this genus, A. psychrotrophicus and A. pellizariae were attributed with potential biotechnological applications 16 . A. psychrotrophicus produced an antifreeze protein 94 , presented hydrocarbon biodegradation activity 95 and showed antitumoral and antiprotozoal activity 96 . In addition, agar-block assays with A. psychrotrophicus described that this fungi has low antibacterial potential against E. coli, showing an inhibition growth zone between 7-10 mm 97 . On the other hand A. pellizariae produced a blue pigment with potential use in the food industry 61 .
To screen for bioactivity, this study used a low-cost in vitro assay adapted to the low temperature growth requirement of the fungi and the high temperature requirement for bacterial growth. This quick assay allowed us to detect bacterial growth inhibition zones around the fungi plugs as indicative of potential antibacterial activity. Without a complete knowledge of the environmental and nutrient requirements for the Antarctic fungi to produce bioactive compounds, we believe that this bioassay has its merit in detecting potential antibacterial metabolites that would have been missed otherwise. This bioassay may be extended to screen for antiviral and anticancer compounds, as well. Future studies will aim to isolate, identify, and characterize the putative bioactive compound(s). This work contributes to the preliminary description of soil fungi of Antarctica and to underscore its potential biotechnological applications and, thus, the importance of its environment conservation.

Material and methods
Soil sampling. The fungi evaluated in this study were isolated from soil samples collected in the Antarctic summer of 2008, near the "Pedro Vicente Maldonado" Antarctic Ecuadorian Scientific Station, located in Fort William Point, Greenwich Island. A total of three sites (stations GIT4-200, GIT4-400, and GIT4-1K) were sampled along a 1000 m linear transect (Fig. 5). At each sampling site, five soil sample replicates were collected with a sterile scoop in a 5 m radius from the registered GPS coordinate. The first 10 cm of soil surface from these five replicates were pooled and filtered with a 2 mm mesh. Soil samples were sealed in sterile polyethylene Amplification and cloning of the ITS region. Antibiotic susceptibility tests. The clinical bacterial isolates used for the fungi antibacterial activity were previously diagnosed by classical antibiotic susceptibility tests using the Kirby Bauer method with the Agar Muller-Hinton media (Thermo Scientific™). Because the Antarctic fungi were grown at low temperature and it is unknown the environmental conditions that may affect their potential antimicrobial activity, we performed the in vitro antibacterial assays on PDA plates at 4 °C and 37 °C. To this end, we first tested if an antibiotic resistant clinical isolate of Klebsiella pneumoniae was able to grow on PDA at 4 °C and 37 °C and still show antibiotic resistance. Bacteria streaked on PDA and Luria broth agar (LBA) were grown at 4 °C and 37 °C. The plates grown at 37 °C were incubated for 24 h, but the plates grown at 4 °C were incubated for 5 days, then the plates were transfer to 37 °C and incubated for further 24 h. On each plate antibiotic disks impregnated with 10 ug of imipenem and meropenem were deposited. Antibiotic resistance, depicted as clear rings around the antibiotic disks, were read after the 37 °C incubation in all treatments.
Assay of antibacterial potential. The antibacterial potential for the fungi was determined using the mycelia plugs method 106 , with fungi isolates grown at 4 °C on PDA. The fungi were analyzed at three sampling times of growth i.e. 15, 30, and 60 days. The clinical bacterial strains used in this assay were: Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis and Staphylococcus aureus. Plates with mycelia plugs and bacterial lawn were first incubated at 4 °C for 5 days on PDA to allow for fungi to grow and then transferred to 37 °C for 24 h for bacterial growth. The bioassays were performed with a minimum of three replicates, and the mean inhibition zone was calculated by measuring the border of the fungi colony to the border of the bacterial growth. This was photographed and measured in millimeters (mm) using the Motic Images Plus 2.0 software. The software SPSS 19 was used for the statistical analysis of the bacterial inhibition zone around the mycelia plug. The